Nucleic acids encoding antibodies having altered effector function and methods for making the same

ABSTRACT

The invention provides a method of producing aglycosylated Fc-containing polypeptides, such as antibodies, having desired effector function. The invention also provides aglycosylated antibodies produced according to the method as well as methods of using such antibodies as therapeutics.

RELATED INFORMATION

This application is a divisional of U.S. application Ser. No.11/360,938, filed Feb. 22, 2006, which is a continuation of co-pendingInternational Application No. PCT/US2004/027476, Filed Aug. 23, 2004,which, in turn, claims priority to U.S. provisional patent applicationNo. 60/497,193, filed on Aug. 22, 2003. The entire contents of theabove-identified applications are hereby incorporated by reference intheir entirety.

The contents of any patents, patent applications, and references citedthroughout this specification are hereby incorporated by reference intheir entireties.

BACKGROUND OF THE INVENTION

The immune response is a mechanism by which the body defends itselfagainst foreign substances that invade it, causing infection or disease.This mechanism is based on the ability of antibodies produced oradministered to the host to bind the antigen though its variable region.Once the antigen is bound by the antibody, the antigen is targeted fordestruction, often mediated in part, by the constant region or Fc domainof the antibody.

For example, one activity of the Fc domain of the antibody is to bindcomplement proteins which can assist in lysing the target antigen, forexample, a cellular pathogen. Another activity of the Fc region is tobind to Fc receptors (FcR) on the surface of immune cells, or so-calledeffector cells, which have the ability to trigger other immune effects.These immune effects include, for example, release of immune activators,regulation of antibody production, endocytosis, phagocytosis, and cellkilling. In some clinical applications these responses are crucial forthe efficacy of the antibody while in other cases they provoke unwantedside effects. One example of an effector-mediated side effect is therelease of inflammatory cytokines causing an acute fever reaction.Another example is the long term deletion of antigen-bearing cells.

The effector function of an antibody can be avoided by using antibodyfragments lacking the Fc region (e.g., such as a Fab, Fab′2, or singlechain antibody (sFv)) however these fragments have a reduced half-life,only one antigen binding site instead of two (e.g., in the case of Fabantibody fragments and single chain antibodies (sFv)), and are moredifficult to purify.

Currently there are limited ways to reduce the effector function of anantibody while retaining the other valuable attributes of the Fc region.One approach is to mutate amino acids on the surface of the antibodythat are involved in the effector binding interactions. While somemutations lead to a reduction of effector function, residual activityusually remains. Moreover, these added mutations can make the antibodyimmunogenic.

Another approach to reduce effector function is to remove sugars thatare linked to particular residues in the Fc region, by for example,deleting or altering the residue the sugar is attached to, removing thesugars enzymatically, by producing the antibody in cells cultured in thepresence of a glycosylation inhibitor, or by expressing the antibody incells unable to glycosylate proteins. However, the forgoing approachesleave residual effector function both in the form ofcomplement-dependent cytolytic activity and Fc receptor binding. Thus, afurther decrease in effector function would be important to guaranteecomplete ablation of activity.

Accordingly, a need exists for an improved method of makingaglycosylated antibodies with altered or reduced effector function.

SUMMARY OF THE INVENTION

The invention solves the foregoing problems of glycosylated antibodies,indeed of any Fc-containing protein, by providing improved methods forproducing aglycosylated antigen binding proteins, for example,aglycosylated antibodies, more specifically, aglycosylated IgGantibodies, by introducing only minimal alterations. In particular, theinvention provides a method for introducing an amino acid alteration ata first amino acid residue position which results in the reducedglycosylation of the polypeptide at a different or second amino acidresidue position. The first amino acid can be modified to comprise adesirable side chain chemistry such that it can be linked, for example,to an additional functional moiety, such as a blocking moiety,detectable moiety, diagnostic moiety, or therapeutic moiety. Theresulting aglycosylated antigen binding polypeptides, for example,aglycosylated IgG antibody has, for example, altered or reduced effectorfunction. The decrease in undesired effector function provided by thepolypeptides and methods of the invention was surprisingly moresubstantial than other conventional means of aglycosylating Fc regions.

Accordingly, the invention has several advantages which include, but arenot limited to, the following:

providing aglycosylated antigen binding polypeptides, for example,aglycosylated IgG antibodies, suitable as therapeutics because of theirreduced effector function;

an efficient method of producing aglycosylated antibodies with minimalalterations to the polypeptide;

an efficient method of producing aglycosylated antibodies while alsoproviding a site for linking a desirable functional moiety, such as ablocking moiety, detectable moiety, diagnostic moiety, or therapeuticmoiety;

a method of altering the effector function of an antibody while avoidingany increase in immunogenicity; and

methods for treating a subject in need of an aglycosylated antigenbinding polypeptide therapy.

Accordingly, in one aspect, the invention provides a polypeptide, orvariant polypeptide, containing an Fc region, wherein the Fc region hasa modified first amino acid residue having a preferred side chainchemistry, and a second amino acid residue having reduced glycosylationas compared to an unmodified polypeptide or parent polypeptide.

In certain embodiments, the side chain chemistry of the first amino acidresidue can be linked, for example, covalently linked, to an additionalmoiety, i.e., a functional moiety such as, for example, a blockingmoiety, detectable moiety, diagnostic moiety, and/or therapeutic moiety.

In one embodiment, the functional moiety is a blocking moiety, in thatthe moiety inhibits or blocks glycosylation of the polypeptide at thesecond amino acid residue. The blocking moiety can also function toblock effector function, for example, by inhibiting the binding of theFc region of the polypeptide to an Fc receptor or complement protein.

In a preferred embodiment, the blocking moiety is a cysteine adductwhich forms when the first amino acid residue is a cysteine or has aside chain chemistry comprising a thiol.

In certain embodiments, the first amino acid comprises a cysteine,cysteine adduct, cystine, mixed disulfide adduct, or disulfide linkage.

In another preferred embodiment, the blocking moiety is a polyalkyleneglycol moiety, for example, a PEG moiety and preferably a PEG-maleimidemoiety.

In a related embodiment, to the first amino acid of the polypeptide is acysteine or has a side chain chemistry comprising a thiol and the PEGmoiety is attached thereto.

In certain embodiments, the cysteine or thiol side chain chemistry isreduced to remove such cysteine adduct, cystine, mixed disulfide adduct,or disulfide linkage, and the PEG moiety is subsequently attached to thecysteine residue or thiol side chain.

In another embodiment, the functional moiety is a detectable moiety,such as, but not limited to, a fluorescent moiety or isotopic moiety.

In another embodiment, the functional moiety is a diagnostic moiety,which is a moiety capable of revealing the presence of a disease ordisorder.

In another embodiment, the functional moiety is a therapeutic moietysuch as, but not limited to, an anti-inflammatory agent, anti-canceragent, anti-neurodegenerative agent, or anti-infective agent.

In another aspect, the variant polypeptide of a parent polypeptidecomprises an Fc region with a modified first amino acid residue, whereinthe modified first amino acid is spatially positioned such that reducedglycosylation at a second amino acid is achieved. In a preferredembodiment, the variant polypeptide, which is aglycosylated, also hasreduced effector function, as compared to the parent polypeptide.

In a related embodiment, the modified first amino acid is spatiallypositioned from the second amino acid by an interval of at least 1 aminoacid position or more, for example, by about 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acid residue positions or more.

In one embodiment, the modified first amino acid residue has a preferredside chain chemistry. In a related embodiment, the preferred side chainchemistry is of sufficient steric bulk and/or charge such that thepolypeptide displays reduced glycosylation and/or effector function.

In one embodiment, the reduced effector function is reduced binding toan Fc receptor (FcR), such as FcγRI, FcγRII, FcγRIII, and/or FcγRIIIb.

In another embodiment, the reduced effector function is reduced bindingto a complement protein, such as C1q.

In a related embodiment, the reduced binding is by a factor of about1-fold to about 15-fold or more.

In another embodiment, the polypeptide has a first amino acid residueand second amino acid residue that are near or within a glycosylationmotif, for example, an N-linked glycosylation motif that contains theamino acid sequence NXT or NXS. In a particular embodiment, thepolypeptide of the method has a first amino acid residue modified by anamino acid substitution. In a related embodiment, the first amino acidresidue is amino acid 299 and the second amino acid residue is aminoacid 297, according to the Kabat numbering.

In another embodiment, the amino acid substitution is selected from thegroup consisting of T299A, T299N, T299G, T299Y, T299C, T299H, T299E,T299D, T299K, T299R, T299G, T299I, T299L, T299M, T299F, T299P, T299W,and T299V according to the Kabat numbering.

In a particular embodiment, the amino acid substitution is T299C orT299A.

In another embodiment, the polypeptide of the invention is pegylated atthe modified first amino acid residue, for example, a cysteine residue,and in particular, with PEG-maleimide.

In a preferred embodiment, the polypeptide is an antibody, for example,an antibody having an Fc region obtained from an antibody such as IgG1,IgG2, IgG3, or IgG4, and preferably, IgG1 or IgG4.

In yet another embodiment, the foregoing polypeptide displays alteredeffector function, for example, reduced binding to an Fc receptor (FcR)(such as FcγRI, FcγRII, or FcγRIII) or reduced binding to a complementprotein, such as C1q.

In another embodiment, the forgoing polypeptide binds to an antigen suchas a ligand, cytokine, receptor, cell surface antigen, or cancer cellantigen.

In another embodiment, the foregoing polypeptide is in a suitablepharmaceutical carrier.

In a another aspect, the invention provides an isolated nucleic acidencoding any one of the foregoing polypeptides, wherein the nucleic acidcan be encoded in a vector, such that, for example, the nucleic acid orvector encoding the same can be expressed in a host cell.

In a another aspect, the invention provides a method for producing anantigen binding polypeptide by culturing the foregoing host cellcontaining a nucleic acid encoding a polypeptide of the invention undersuitable culture conditions for producing the polypeptide followed by,for example, recovering the polypeptide from the host cell culture.

In a another aspect, the invention provides a method of producing amodified antigen binding polypeptide having reduced glycosylation in anFc region, by identifying an original first amino acid residue in anoriginal polypeptide and a second amino acid residue capable of beingglycosylated in an Fc region of the original polypeptide, and modifyingthe original first amino acid residue in the original polypeptide toproduce a modified first amino acid in a modified polypeptide, such thatglycosylation of the second amino acid residue of the Fc region isdecreased in the modified or variant polypeptide as compared to theoriginal or parent polypeptide.

In one embodiment, the method can comprise the step of determining ifthe modified antigen binding polypeptide displays altered effectorfunction.

In another aspect, the invention provides a method of reducing effectorfunction by identifying a first amino acid residue in the antibody,which when modified, is capable of altering the glycosylation of thesecond amino acid residue in the Fc region of the antibody. Theidentifying of the first amino acid residue to be modified can becomputer-assisted using, for example, art recognized modeling software.The first amino acid residue is then modified such that glycosylation ofthe second amino acid residue of the Fc region is reduced in themodified antibody as compared to the unmodified parent antibody.

In another aspect, the invention provides a polypeptide produced by anyone of the foregoing methods.

In another aspect, the invention provides a method of diagnosing,treating, or preventing a disease or disorder in an animal, for example,a human patient, by administering a polypeptide of the invention havingreduced glycosylation and/or effector function.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of a typical antigen binding polypeptide(IgG antibody) and the functional properties of antigen binding andeffector function (e.g., Fc receptor (FcR) binding) of an antibody. Alsoshown is how the presence of sugars (glycosylation) in the CH2 domain ofthe antibody alters effector function (FcR binding) but does not affectantigen binding.

FIG. 2 depicts the structure and sequence of an Fc region of an antibodyof the invention where a residue proximal to the glycosylated amino acidreside can be altered to inhibit glycosylation (left panel). Also shown(right panel) is that if the first amino acid reside is a cysteine,glycosylation is not only inhibited but the cysteine residue provides asite for linking a functional moiety, e.g., a blocking moiety, such as acysteine adduct or pegylation moiety (shown) or other functionalmoieties (not shown).

FIG. 3 depicts a digital image of SDS-PAGE analysis of glycosylatedantibodies and aglycosylated antibody IgG1 variants under non-reducingconditions (lanes 1-5) and reducing conditions (lanes 7-11). Theaglycosylated antibody variants (or Fc regions thereof) migrate fasterthan glycosylated controls because they lack the added sugar moieties(compare lanes 3-5 with lane 2 and lanes 9-11 with lane 8). Inparticular, lane 1 contains a control full length antibody (monoclonalIgG1), lane 2 contains a control wild type (glycosylated) Fc region(IgG1), lane 3 contains an aglycosylated Fc variant (N297Q human IgG1),lane 4 contains an aglycosylated Fc variant (T299A human IgG1), lane 5contains an aglycosylated Fc variant (T299C human IgG1), lane 6 containsmolecular weight standards, lane 7 contains a control full lengthantibody (monoclonal IgG1), lane 8, contains a control wild type(glycosylated) Fc region (IgG1), lane 9 contains an aglycosylated Fcvariant (N297Q human IgG1), lane 10 contains an aglycosylated Fc variant(T299A human IgG1), and lane 11 contains an aglycosylated Fc variant(T299C human IgG1).

FIG. 4 depicts a digital image of SDS-PAGE analysis of glycosylatedantibodies and aglycosyl antibody IgG4 variants under non-reducingconditions (lanes 1-3) and reducing conditions (lanes 5-7). The IgG4aglycosyl antibody variant migrates faster than the glycosylated controlbecause it lacks the added sugar moieties (compare lane 3 with lane 2and lane 7 with lane 6). In particular, lanes 1 and 5 contain a controlIgG1, lanes 2 and 6 contain a control IgG4 antibody, and lanes 3 and 7contain the IgG4 aglycosyl variant (T299A). Lane 4 contains molecularweight standards.

FIG. 5 depicts a digital image of SDS-PAGE analysis of aglycosylatedantibody variants (Fc regions) under non-reducing conditions showingthat cysteines are blocked in the presence (lanes 3, 4, 8, and 9) orabsence (lanes 1, 2, 6, and 7) of peg-maleimide. In particular, lanes1-4 contain T299C and lanes 6-9 contain T299A, with molecular weightstandards in lane 10.

FIG. 6 depicts a digital image of SDS-PAGE analysis of aglycosylatedantibody variants (Fc regions) under reducing conditions showing thatintroduced cysteines (T299C) are pegylated but alanine resides (T299A)are not, as evidenced by reduced mobility. In particular, lanes 1-2 wereloaded with increasing amounts (2.5 ug, 7.5 ug) of Fc T299C, lanes 3-4were loaded with pegylated Fc T299C, lanes 5-6 were loaded withincreasing amounts of Fc T299A, lanes 7-8 were loaded with pegylated FcT299A, and lane 9 was loaded with a protein molecular weight marker.

FIG. 7 depicts a digital image of SDS-PAGE analysis of the pegylation ofthe antibody variant T299C (Fc region) as compared to antibody variantT299A (Fc region) under non-reducing and non-denaturing conditions afterfirst reducing the test proteins with TCEP to remove the cysteine adductfollowed by pegylation showing that the introduced cysteines (T299C) arepegylated but alanine resides (T299A) are not, as evidenced by reducedmobility. In particular, lane 1 was loaded with Fc T299A after reductionand reoxidation, non-reducing gel conditions, lane 2 with Fc T299C afterreduction and reoxidation, non-reducing gel conditions, lane 3 with aprotein molecular weight marker, lane 4 Fc T299A with no peg-maleimide,reducing gel conditions, lane 5 Fc T299C no peg-maleimide, reducing gelconditions, lane 6 Fc T299A plus peg-maleimide, reducing gel conditions,and lane 7 with Fc T299C plus peg-maleimide, reducing gel conditions.

FIGS. 8-11 show mass spectroscopy histogram analyses of aglycosylatedantibody variants having cysteine (T299C) or alanine (T299A) mutationsunder reducing and non-reducing conditions. The mass spectroscopy datashows that under non-reducing conditions the T299C antibody variant hasadded mass due to the formation of a cysteine adduct coupled to thecysteine at position 299 but that such an adduct does not form when analanine is present (i.e., T299A).

FIG. 12 shows the decreased effector function of the aglycosylatedantibody IgG1 variants of the invention as a function of FcγRI (upperpanel) or FcγRIII (lower) binding. The T299C variant, which is bothaglycosylated and modified by a cysteine adduct, has less effectorfunction (FcγRI binding) as compared to merely aglycosylated antibodies(upper panel).

FIG. 13 shows the decreased effector function of the aglycosylatedantibody IgG4 variant of the invention as a function of FcγRI (upperpanel) or FcγRIII (lower) binding. The T299A IgG4 variant has lesseffector function (FcγRI binding) as compared to the aglycosylated IgG1form.

FIG. 14 shows the decreased effector function of the aglycosyl IgG1antibody (i.e., hu5c8) as a function of binding to the complementprotein C1q. The T299C variant, which is both aglycosylated and modifiedby a cysteine adduct, has less effector function (i.e., C1q binding) ascompared to the aglycosylated only form.

FIG. 15 shows the decreased effector function of the aglycosyl IgG4antibody (i.e., hu5c8) as a function of binding to the complementprotein C1q. The T299A IgG4 variant has less effector function (i.e.,C1q binding) as compared to the aglycosylated IgG1 variant.

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear understanding of the specification andclaims, the following definitions are conveniently provided below.

Definitions

The term “antibody” includes monoclonal antibodies (including fulllength monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), chimeric antibodies,CDR-grafted antibodies, humanized antibodies, human antibodies, andfragments thereof where reduced glycosylation and/or effector functionis desirable, for example, an antibody light chain (VL), an antibodyheavy chain (VH), a single chain antibody (scFv), a F(ab′)2 fragment, aFab fragment, an Fd fragment, an Fv fragment, and a single domainantibody fragment (DAb).

The term “parent antibody” includes any antibody for which modificationof the glycosylation, effector function, and/or the providing of apreferred or desirable side chain chemistry for adding, for example, afunctional moiety, is desired. Thus, the parent antibody represents theoriginal antibody on which the methods of the instant invention areperformed. The parent polypeptide may comprise a native sequence (i.e. anaturally occurring) antibody (including a naturally occurring allelicvariant), or an antibody with pre-existing amino acid sequencemodifications (such as insertions, deletions and/or other alterations)of a naturally occurring sequence. The parent antibody may be amonoclonal, chimeric, CDR-grafted, humanized, or human antibody.

The terms “antibody variant” or “modified antibody”, includes anantibody which has an amino acid sequence or amino acid side chainchemistry which differs from that of the parent antibody by at least oneamino acid or amino acid modification as described herein. In preferredembodiments, the antibody variant will have reduced glycosylation, and,optionally, reduced effector function as compared to the parent antibodyand/or further comprise one or more functional moieties.

The term “first amino acid residue” refers to the amino acid residue (orposition) of the polypeptide which is modified by the insertion,substitution, or deletion of an amino acid residue or by directlyaltering the side chain chemistry of the existing amino acid residue,such that the modified amino acid residue (or residue position) isdifferent and thereby reduces or eliminates glycosylation of a secondamino acid residue. Preferably, the modification of the first aminoacid, while influencing the glycosylation and/or effector function ofthe polypeptide (and optionally providing a site for linking afunctional moiety), the modification does not significantly alter otherdesired functions of the polypeptide nor does the functional moietyattached thereto. For example, where the Fc containing polypeptide is anantibody, the modification of the first amino acid does notsignificantly alter the antigen-binding activity of the antibody.

The term “second amino acid residue” refers to the amino acid residue ofthe polypeptide which is capable of being covalently linked to one ormore carbohydrates, for example, glycosylated.

The term “preferred side chain chemistry” refers to a chemistry, forexample, an amino acid residue side chain or R-group chemistry thatimparts a desirable characteristic to the polypeptide. The preferredside chain chemistry is introduced at the first amino acid position byamino acid substitution, by chemical substitution such that its sidechain chemistry is modified, or by an amino acid addition or deletionsuch that a different amino acid side chain chemistry is provided at thefirst amino acid position. As described herein, modification of the sidechain chemistry of the parent antibody so that it contains the preferredside chain chemistry reduces glycosylation at a second amino acidposition, resulting in reduced effector function. The modification alsoprovides a site for linking a desirable functional moiety. In certainembodiments, a determination as to the preferred side chain chemistrymay be informed by an in silico or computer-based approach fordetermining the steric bulk, and/or charge of the side chain chemistryto be introduced (e.g., by substitution) at the first amino acidposition.

The term “amino acid” includes alanine (Ala or A); arginine (Arg or R);asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C);glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G);histidine (His or H); isoleucine (Ile or I): leucine (Leu or L); lysine(Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline(Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp orW); tyrosine (Tyr or Y); and valine (Val or V). Non-traditional aminoacids are also within the scope of the invention and include norleucine,ornithine, norvaline, homoserine, and other amino acid residue analoguessuch as those described in Ellman et al. Meth. Enzym. 202:301-336(1991). To generate such non-naturally occurring amino acid residues,the procedures of Noren et al. Science 244:182 (1989) and Ellman et al.,supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.Introduction of the non-traditional amino acid can also be achievedusing peptide chemistries know in the art.

The term “preferred side chain chemistry is of sufficient steric bulk”includes the side chain chemistry of an amino acid residue havingsufficient steric bulk so as to inhibit the glycosylation of an Fccontaining polypeptide and/or its effector function. Such residuesinclude, for example, phenylalanine, tyrosine, tryptophan, arginine,lysine, histidine, glutamic acid, glutamine, and methionine, or analogsor mimetics thereof.

The term “preferred side chain chemistry is of sufficient charge” or“electrostatic charge” includes the side chain chemistry of an aminoacid residue having sufficient charge so as to inhibit the glycosylationof an Fc containing polypeptide and/or its effector function. Suchresidues include, for example, the negatively charged amino acidresidues, e.g., aspartic acid, glutamic acid, or analogs or mimeticsthereof, and the positively charged amino acid residues, e.g., lysine,arginine, histidine, and analogs or mimetics thereof.

The term “preferred side chain chemistry is of sufficient steric bulkand charge” includes the side chain chemistry of an amino acid residuehaving sufficient steric bulk and charge so as to inhibit theglycosylation of an Fc containing polypeptide and/or its effectorfunction. Such residues include, for example, lysine, arginine,tyrosine, and analogs or mimetics thereof.

The term “sufficient” as used herein, generally refers to the preferredmodifications described herein which achieve at least one of thefollowing in an Fc containing polypeptide: reduced glycosylation of thepolypeptide; reduced effector function of the polypeptide; and/orproviding of a site for linking a functional moiety.

The term “functional moiety” includes moieties which, preferably, add adesirable function to the variant polypeptide. Preferably, the functionis added without significantly altering an intrinsic desirable activityof the polypeptide, e.g., in the case of an antibody, theantigen-binding activity of the molecule. A variant polypeptide of theinvention may comprise one or more functional moieties, which may be thesame or different. Examples of useful functional moieties include, butare not limited to, a blocking moiety, a detectable moiety, a diagnosticmoiety, and a therapeutic moiety. Exemplary blocking moieties includemoieties of sufficient steric bulk and/or charge such that reducedglycosylation occurs, for example, by blocking the ability of aglycosidase to glycosylate the polypeptide. The blocking moiety mayadditionally or alternatively, reduce effector function, for example, byinhibiting the ability of the Fc region to bind a receptor or complementprotein. Preferred blocking moieties include cysteine adducts, cystine,mixed disulfide adducts, and PEG moieties. Exemplary detectable moietiesinclude fluorescent moieties, radioisotopic moieties, radiopaquemoieties, and the like. Exemplary diagnostic moieties include moietiessuitable for revealing the presence of an indicator of a disease ordisorder. Exemplary therapeutic moieties include, for example,anti-inflammatory agents, anti-cancer agents, anti-neurodegenerativeagents, and anti-infective agents. The functional moiety may also haveone or more of the above-mentioned functions. Other useful functionalmoieties are known in the art and described, below.

The term “pegylation”, “polyethylene glycol”, or “PEG” includes apolyalkylene glycol compound or a derivative thereof, with or withoutcoupling agents or derivatization with coupling or activating moieties(e.g., with thiol, triflate, tresylate, azirdine, oxirane, or preferablywith a maleimide moiety, e.g., PEG-maleimide). Other appropriatepolyalkylene glycol compounds include, but are not limited to, maleimidomonomethoxy PEG, activated PEG polypropylene glycol, but also charged orneutral polymers of the following types: dextran, colominic acids, orother carbohydrate based polymers, polymers of amino acids, and biotinderivatives.

The term “spatially positioned” includes the relative position ordistance between the modified first amino acid position and the secondamino acid position within a polypeptide where it is desirable to alteror reduce the glycosylation at the second amino acid position bymodifying the first amino acid position. Amino acid distances of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-20 or more amino acid positions, orany interval of the foregoing ranges are within the scope of theinvention. Methods of determining that the desired spatial positioningof the first and second amino acids achieves the desired effect, forexample, reduced glycosylation and/or effector function, are known inthe art and are described herein (see, e.g., Examples 1 and 4).

The term “effector function” refers to the functional ability of the Fcor constant region of an antibody to bind proteins and/or cells of theimmune system. Typical effector functions include the ability to bindcomplement protein (e.g., the complement protein C1q), and/or an Fcreceptor (FcR) (e.g., FcγRI, FcγRII, FcγRIII, and/or FcγRIIIb). Thefunctional consequences of being able to bind one or more of theforegoing include opsonization, phagocytosis, antigen-dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and/oreffector cell modulation. A decrease in effector function refers to adecrease in one or more of the biochemical or cellular activities, whilemaintaining the antigen binding activity of the variable region of theantibody (or fragment thereof). Decreases in effector function, e.g., Fcbinding to an Fc receptor or complement protein, can be expressed interms of fold reduction (e.g., reduced by 1-fold, 2-fold, and the like)and can be calculated based on, e.g., the percent reductions in bindingactivity determined using the assays described herein (see, e.g.,Example 4) or assays known in the art.

The term “glycosylation” refers to the covalent linking of one or morecarbohydrates to a polypeptide. Typically, glycosylation is aposttranslational event which can occur within the intracellular milieuof a cell or extract therefrom. The term glycosylation includes, forexample, N-linked glycosylation (where one or more sugars are linked toan asparagine residue) and/or O-linked glycosylation (where one or moresugars are linked to an amino acid residue having a hydroxyl group(e.g., serine or threonine).

All amino acid numberings herein for an Fc region of a polypeptidecorrespond to the Kabat numbering system as described, e.g., by Kabat etal., in “Sequences of Proteins of Immunological Interest”, U.S. Dept.Health and Human Services, 1983 and 1987.

DETAILED DESCRIPTION

A method has been developed to produce aglycosylated antigen-bindingpolypeptides, for example, antibodies or Fc-containing fusion proteins,by altering a first amino acid residue that inhibits the glycosylationat a second amino acid residue. The method is especially well suited forproducing therapeutic aglycosylated Fc-containing polypeptides ineukaryotic cells with only minimal amino acid alterations to thepolypeptide. The methods of the present invention thereby avoidsintroducing into the polypeptide amino acid sequence that can beimmunogenic.

Preferably, the modification of the first amino acid, while influencingthe glycosylation and/or effector function of the polypeptide (andoptionally providing a site for linking a functional moiety), does notsignificantly alter other desired functions of the polypeptide nor doesthe functional moiety attached thereto. For example, where the Fccontaining polypeptide is an antibody, the modification of the firstamino acid does not significantly alter the antigen-binding activity ofthe antibody.

Accordingly, the method is suitable for producing therapeuticantibodies, for example, IgG antibodies, where altered or reducedeffector function is desired. The altered or reduced effector functionis achieved by reducing or eliminating the glycosylation of the Fcregion of the antibody using the method of the invention (FIG. 1). Inparticular, a first amino acid residue(s) is targeted for alteration(e.g., by substitution, insertion, deletion, or by chemicalmodification) which inhibits the glycosylation of a second amino acidresidue. The resultant antibody is aglycosylated at the second aminoacid residue and has altered or reduced effector function, e.g.,complement binding activity or effector cell activity such as binding toan Fc receptor.

In certain embodiments, the reduced effector function is reduced bindingto an Fc receptor (FcR), such as the FcγRI, FcγRII, FcγRIII, and/orFcγRIIIb receptor or a complement protein, for example, the complementprotein C1q. This change in binding can be by a factor of about 1 foldor more, e.g., by about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 50, or 100-foldor more, or by any interval or range thereof.

These decreases in effector function, e.g., Fc binding to an Fc receptoror complement protein, are readily calculated based on, e.g., thepercent reductions in binding activity determined using the assaysdescribed herein (see, e.g., Example 4) or assays known in the art.

In another embodiment, the first amino acid residue is modified orsubstituted to contain a preferred side chain chemistry of sufficientsteric bulk and/or charge such that reduced glycosylation and oreffector function is achieved.

Exemplary amino acid residues having side chain chemistry of sufficientsteric bulk include phenylalanine, tyrosine, tryptophan, arginine,lysine, histidine, glutamic acid, glutamine, and methionine, or analogsor mimetics thereof.

Exemplary amino acid residues having side chain chemistry of sufficientcharge include, for example, negatively charged amino residues, e.g.,aspartic acid, glutamic acid analogs or mimetics thereof, and positivelycharged amino acid residues, e.g., lysine, arginine, histidine, andanalogs or mimetics thereof.

Further, amino acid residues that are uncharged at physiological pH maybecome charged when residing in an environment that alters thephysiological pH, e.g., serine, threonine, cysteine, methionine,asparagine, glutamine, tyrosine, and analogs or mimetics thereof. Forexample, uncharged amino acid residues can be buried inside a foldedprotein and experience a shift in pKa, thereby altering the charge ofthe residue compared to the charge at physiological pH.

In one embodiment, the preferred amino acid residue is of sufficientsteric bulk and charge such that the residue inhibits glycosylation at asecond amino acid position. Such amino acids include, for example,lysine, arginine, and tyrosine.

In preferred embodiments of the present invention, the amino acidresidue that is modified can be selected for additional properties,e.g., to serve as a site for coupling desirable functional moietieswhich impart desirable properties to the polypeptide. Examples of suchpreferred moieties include, e.g., blocking moieties, detectablemoieties, diagnostic moieties, and therapeutic moieties.

In another embodiment, the variant polypeptide of a parent polypeptidecontains an Fc region, which comprises a modified first amino acidresidue, wherein the modified first amino acid is spatially positionedsuch that reduced glycosylation at a second amino acid is achieved,whereby the variant polypeptide has reduced effector function ascompared to the parent polypeptide.

Preferred spatial positioning can be based on the predicted proximity ofthe first amino acid to the second amino acid as well as the steric bulkand/or charge of the preferred side chain chemistry to be introduced atthe first amino acid position. Alternatively, a determination as to theoptimal spatial positioning may be informed by empirical observationsafter substitutions of a preferred amino acid side chain chemistry atone or more positions and/or using an art recognized in silico orcomputer-based approach for determining the steric bulk, charge, and/orthe distance of the first amino acid position from the second amino acidposition. Amino acid distances of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20 or more residue positions, or any interval of the foregoingranges, are within the scope of the invention. Thus, in certainpreferred embodiments, the modified first amino acid is spatiallypositioned from the second amino acid by an interval of at least 1 aminoacid position or more, for example, by 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acid positions or more.

Methods of determining that the desired spatial positioning of the firstand second amino acids achieves the desired effect, for example, reducedglycosylation and/or effector function, are described herein (see, e.g.,Examples 1 and 4).

In a preferred embodiment, the polypeptide of the invention is an Fccontaining polypeptide such as an antibody, and preferably IgGimmunoglobulin, e.g., of the subtype IgG1, IgG2, IgG3, or IgG4, andpreferably, of the subtype IgG1 or IgG4. In a preferred embodiment, theforgoing polypeptide binds to an antigen such as a ligand, cytokine,receptor, cell surface antigen, or cancer cell antigen.

Because the invention provides an isolated nucleic acid encoding any oneof the foregoing polypeptides, the nucleic acid can be introduced into avector and expressed in a host cell. Accordingly, a polypeptide of theinvention can be produced by culturing a suitable host cell containing anucleic acid encoding a polypeptide of the invention under appropriateculture conditions for producing the polypeptide.

In a preferred embodiment, the polypeptide of the invention has a firstamino acid that has been modified to have a cysteine residue or sidechain chemistry thereof, i.e., a thiol, such that the polypeptide, underthe above culture conditions, is capable of forming an adduct with afree cysteine provided under the culture conditions. In a preferredembodiment, the resulting polypeptide has reduced glycosylation andeffector function.

In a related embodiment, the polypeptide can be further manipulated, forexample, subjected to reducing conditions, such that the cysteineadduct, cystine, mixed disulfide adduct, or disulfide linkage, isremoved thereby providing a site for further modifying the polypeptidewith a functional moiety, for example, a pegylation moiety.

In another embodiment, the polypeptide has a first amino acid residueand second amino acid residue that are near or within a glycosylationmotif, for example, an N-linked glycosylation motif that contains theamino acid sequence NXT or NXS. In a particular embodiment, thepolypeptide of the method has a first amino acid residue modified by anamino acid substitution. In a related embodiment, the first amino acidresidue is amino acid 299 and the second amino acid residue is aminoacid 297, according to the Kabat numbering.

In another embodiment, the amino acid substitution is selected from thegroup consisting of T299A, T299N, T299G, T299Y, T299C, T299H, T299E,T299D, T299K, T299R, T299G, T299I, T299L, T299M, T299F, T299P, T299W,and T299V according to the Kabat numbering.

In a particular embodiment, the amino acid substitution is T299C orT299A.

Although the method of the invention described herein uses an IgGantibody that is normally N-glycosylated at a particular residue in theFc region (amino acid 297) (FIGS. 1-2), it is understood that the methodcan be equally applied to an Fc region within any polypeptide. When thepolypeptide is an antibody, the antibody can be synthetic,naturally-derived (e.g., from serum), produced by a cell line (e.g., ahybridoma), or produced in a transgenic organism. Still further, themethod may also be applied to a polypeptide which does not comprise anFc region provided the polypeptide comprises at least one glycosylationsite.

The method offers several advantages over current mutagenesis methods,for example, because the method can be used to inhibit glycosylation ofthe polypeptide in a way that is minimally disruptive (FIG. 2, leftpanel), e.g., without the mutation of the normally glycosylated residue,deletion of the glycosylation site, or enzymatic removal of the sugarmoieties. Accordingly, the structure of the polypeptide is maintained,the binding affinity of the polypeptide for antigen is maintained,immunogenicity of the polypeptide is avoided, and the polypeptide canbe, if desired, coupled to a desirable functional moiety (FIG. 2, rightpanel). Such functional moieties can further abrogate effector functionor improve the half-life of the polypeptide or achieve desirabletherapeutic function. Moreover, the methods of the invention can beperformed using standard genetic engineering techniques.

1. Identifying Glycosylation Sites

The method is performed by identifying a glycosylation site in anFc-containing polypeptide, for example, an antibody, in one embodiment,an IgG antibody. The identification of the glycosylation site can beexperimental or based on sequence analysis or modeling data. Consensusmotifs, that is, the amino acid sequence recognized by various glycosyltransferases, have been described. For example, the consensus motif foran N-linked glycosylation motif is frequently NXT or NXS, where X can beany amino acid except proline (FIG. 2). Several algorithms for locatinga potential glycosylation motif have also been described. Accordingly,to identify potential glycosylation sites within an antibody orFc-containing fragment, the sequence of the antibody is examined, forexample, by using publicly available databases such as the websiteprovided by the Center for Biological Sequence Analysis (seewww.cbs.dtu.dk/services/NetNGlyc/ for predicting N-linked glycosylationsites) and www.cbs.dtu.dk/services/NetOGlyc/ for predicting O-linkedglycosylation sites). Additional methods for altering glycosylationsites of antibodies are described, e.g., in U.S. Pat. Nos. 6,350,861 and5,714,350.

In certain cases, the glycosylation of a given motif will depend onother features of the protein, the type of cell or cell extract and theconditions under which the antibody is produced or contacted with such acell or extract. To the extent that a given cell or extract has resultedin the glycosylation of a given motif, art recognized techniques fordetermining if the motif has been glycosylated are available, forexample, using gel electrophoresis and/or mass spectroscopy, asdescribed herein.

Identification of an actual or potential glycosylation motif alsoreveals the residue to which the sugars are covalently linked. Forexample, N-linked glycosylation results in the linking of a sugarresidue (glycan) to the terminal side-chain nitrogen at an asparagineresidue. In another example, O-linked glycosylation results in thecovalent linking of a sugar residue (glycan) to an amino acid residehaving a hydroxyl side group such as serine or threonine. In eithercase, the method of the invention does not alter the residue to whichone or more sugars would be covalently linked. Rather, the method of theinvention employs the alteration of a residue different from the residuewhich would be normally covalently linked to a sugar residue by amechanism that operates in cis thereby inhibiting the coupling of one ormore sugars to the residue but without requiring the alteration of theactual residue capable of being linked to a sugar, i.e., glycosylated.

The methods of the invention are applicable to a variety of usesincluding, the bioproduction of aglycosylated polypeptides usingeukaryotic cells. Such aglycosylated polypeptides, for example,antibodies, are desirable therapeutics for the treatment of humandisease.

2. Production of Antibodies with Altered Fc Regions

Having selected the antibody to be improved, for example, a chimeric,human, humanized, or synthetic antibody, a variety of methods areavailable for producing such antibodies. Because of the degeneracy ofthe code, a variety of nucleic acid sequences will encode each antibodyamino acid sequence. The desired nucleic acid sequences can be producedby de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared polynucleotide encoding the antibody. Oligonucleotide-mediatedmutagenesis is one method for preparing a substitution, deletion, orinsertion of an alteration (e.g., altered codon) that reduces theglycosylation of a second, usually proximal, amino acid. For example,the target polypeptide DNA is altered by hybridizing an oligonucleotideencoding the desired mutation to a single-stranded DNA template. Afterhybridization, a DNA polymerase is used to synthesize an entire secondcomplementary strand of the template that incorporates theoligonucleotide primer, and encodes the selected alteration in thevariant polypeptide-DNA. In one embodiment, genetic engineering, e.g.,primer-based PCR mutagenesis, is sufficient to alter the first aminoacid, as defined herein, for producing a polynucleotide encoding apolypeptide that, when expressed in a eukaryotic cell, will now have anaglycosylated region, for example, aglycosylated Fc region. Theantibodies produced as described above typically comprise at least aportion of an antibody constant region (Fc), typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both light chainand heavy chain constant regions. The heavy chain constant regionusually includes CH1, hinge, CH2, and CH3 regions. It is understood,however, that the antibodies described herein include antibodies havingall types of constant regions, including IgM, IgG, IgD, and IgE, and anyisotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, thehuman isotype IgG1 is used. In another embodiment, the human isotypeIgG4 is used. Light chain constant regions can be lambda or kappa. Thehumanized antibody may comprise sequences from more than one class orisotype. Antibodies can be expressed as tetramers containing two lightand two heavy chains, as separate heavy chains, light chains, as Fab,Fab′ F(ab′)2, and Fv, or as single chain antibodies (sFv) in which heavyand light chain variable domains are linked through a spacer.

Methods for determining the effector function of a polypeptidecomprising an Fc region, for example, an antibody, are described hereinand include cell-based bridging assays to determine changes in theability of a modified Fc region to bind to an Fc receptor. Other bindingassays may be used to determine the ability of an Fc region to bind to acomplement protein, for example, the C1q complement protein. Additionaltechniques for determining the effector function of a modified Fc regionare described in the art.

3. Functional Moieties and the Chemistry of Linking Such Moieties toFc-Containing Polypeptides

The invention provides antibodies and Fc-containing polypeptides thatmay be further modified to provide a desired effect. For example, inpreferred embodiments, the first amino acid is modified to be a residuethat not only alters the glycosylation of the polypeptide at a secondsite, but also provides a desired side chain chemistry.

In certain preferred embodiments, the side chain chemistry of the aminoacid residue is capable of being linked, for example, covalently linked,to an additional moiety, i.e., a functional moiety such as, for example,a blocking moiety, a detectable moiety, a diagnostic moiety, and/or atherapeutic moiety. Exemplary functional moieties are first describedbelow followed by useful chemistries for linking such functionalmoieties to the different amino acid side chain chemistries.

3.1 Functional Moieties

Examples of useful functional moieties include, but are not limited to,a blocking moiety, a detectable moiety, a diagnostic moiety, and atherapeutic moiety.

Exemplary blocking moieties include moieties of sufficient steric bulkand/or charge such that reduced glycosylation occurs, for example, byblocking the ability of a glycosidase to glycosylate the polypeptide.The blocking moiety may additionally or alternatively, reduce effectorfunction, for example, by inhibiting the ability of the Fc region tobind a receptor or complement protein. Preferred blocking moietiesinclude cysteine adducts and PEG moieties.

In a preferred embodiment, the blocking moiety is a cysteine, preferablya cysteine that has associated with a free cysteine, e.g., during orsubsequent to the translation of the Fc containing polypeptide, e.g., incell culture. Other blocking cysteine adducts include cystine, mixeddisulfide adducts, or disulfide linkages.

In another preferred embodiment, the blocking moiety is a polyalkyleneglycol moiety, for example, a PEG moiety and preferably a PEG-maleimidemoiety. Preferred pegylation moieties (or related polymers) can be, forexample, polyethylene glycol (“PEG”), polypropylene glycol (“PPG”),polyoxyethylated glycerol (“POG”) and other polyoxyethylated polyols,polyvinyl alcohol (“PVA) and other polyalkylene oxides, polyoxyethylatedsorbitol, or polyoxyethylated glucose. The polymer can be a homopolymer,a random or block copolymer, a terpolymer based on the monomers listedabove, straight chain or branched, substituted or unsubstituted as longas it has at least one active sulfone moiety. The polymeric portion canbe of any length or molecular weight but these characteristics canaffect the biological properties. Polymer average molecular weightsparticularly useful for decreasing clearance rates in pharmaceuticalapplications are in the range of 2,000 to 35,000 daltons. In addition,if two groups are linked to the polymer, one at each end, the length ofthe polymer can impact upon the effective distance, and other spatialrelationships, between the two groups. Thus, one skilled in the art canvary the length of the polymer to optimize or confer the desiredbiological activity. PEG is useful in biological applications forseveral reasons. PEG typically is clear, colorless, odorless, soluble inwater, stable to heat, inert to many chemical agents, does nothydrolyze, and is nontoxic. Pegylation can improve pharmacokineticperformance of a molecule by increasing the molecule's apparentmolecular weight. The increased apparent molecular weight reduces therate of clearance from the body following subcutaneous or systemicadministration. In many cases, pegylation can decrease antigenicity andimmunogenicity. In addition, pegylation can increase the solubility of abiologically-active molecule.

Pegylated antibodies and antibody fragments may generally be used totreat conditions that may be alleviated or modulated by administrationof the antibodies and antibody fragments described herein. Generally thepegylated aglycosylated antibodies and antibody fragments have increasedhalf-life, as compared to the nonpegylated aglycosylated antibodies andantibody fragments. The pegylated aglycosylated antibodies and antibodyfragments may be employed alone, together, or in combination with otherpharmaceutical compositions.

Examples of detectable moieties which are useful in the methods andpolypeptides of the invention include fluorescent moieties,radioisotopic moieties, radiopaque moieties, and the like, e.g.detectable labels such as biotin, fluorophores, chromophores, spinresonance probes, or radiolabels. Exemplary fluorophores includefluorescent dyes (e.g. fluorescein, rhodamine, and the like) and otherluminescent molecules (e.g. luminal). A fluorophore may beenvironmentally-sensitive such that its fluorescence changes if it islocated close to one or more residues in the modified protein thatundergo structural changes upon binding a substrate (e.g. dansylprobes). Exemplary radiolabels include small molecules containing atomswith one or more low sensitivity nuclei (¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²³I, ⁹⁹Tc,⁴³K, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In and the like). Other useful moieties areknown in the art.

Examples of diagnostic moieties which are useful in the methods andpolypeptides of the invention include detectable moieties suitable forrevealing the presence of a disease or disorder. Typically a diagnosticmoiety allows for determining the presence, absence, or level of amolecule, for example, a target peptide, protein, or proteins, that isassociated with a disease or disorder. Such diagnostics are alsosuitable for prognosing and/or diagnosing a disease or disorder and itsprogression.

Examples of therapeutic moieties which are useful in the methods andpolypeptides of the invention include, for example, anti-inflammatoryagents, anti-cancer agents, anti-neurodegenerative agents, andanti-infective agents. The functional moiety may also have one or moreof the above-mentioned functions.

Exemplary therapeutics include radionuclides with high-energy ionizingradiation that are capable of causing multiple strand breaks in nuclearDNA, and therefore suitable for inducing cell death (e.g., of a cancer).Exemplary high-energy radionuclides include: ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I,¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re. Theseisotopes typically produce high energy α- or β-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells and are essentially non-immunogenic.

Exemplary therapeutics also include cytotoxic agents such as cytostatics(e.g. alkylating agents, DNA synthesis inhibitors, DNA-intercalators orcross-linkers, or DNA-RNA transcription regulators), enzyme inhibitors,gene regulators, cytotoxic nucleosides, tubulin binding agents, hormonesand hormone antagonists, anti-angiogenesis agents, and the like.

Exemplary therapeutics also include alkylating agents such as theanthracycline family of drugs (e.g. adriamycin, carminomycin,cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin,streptonigrin, porfiromycin, anthracenediones, and aziridines). Inanother embodiment, the chemotherapeutic moiety is a cytostatic agentsuch as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitorsinclude, but are not limited to, methotrexate and dichloromethotrexate,3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosineβ-D-arabinofuranoside, 5-fluoro-5′-deoxyuridine, 5-fluorouracil,ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C. ExemplaryDNA-intercalators or cross-linkers include, but are not limited to,bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide,cis-diammineplatinum(II) dichloride (cisplatin), melphalan,mitoxantrone, and oxaliplatin.

Exemplary therapeutics also include transcription regulators such asactinomycin D, daunorubicin, doxorubicin, homoharringtonine, andidarubicin. Other exemplary cytostatic agents that are compatible withthe present invention include ansamycin benzoquinones, quinonoidderivatives (e.g. quinolones, genistein, bactacyclin), busulfan,ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone,indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ,triethylenephosphoramide, and nitrosourea compounds (e.g. carmustine,lomustine, semustine).

Exemplary therapeutics also include cytotoxic nucleosides such as, forexample, adenosine arabinoside, cytarabine, cytosine arabinoside,5-fluorouracil, fludarabine, floxuridine, ftorafur, and6-mercaptopurine; tubulin binding agents such as taxoids (e.g.paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g.Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g. ZD6126),combretastatins (e.g. Combretastatin A-4, AVE-6032), and vinca alkaloids(e.g. vinblastine, vincristine, vindesine, and vinorelbine (navelbine));anti-angiogenesis compounds such as Angiostatin K1-3,DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein,minocycline, staurosporine, and (±)-thalidomide.

Exemplary therapeutics also include hormones and hormone antagonists,such as corticosteroids (e.g. prednisone), progestins (e.g.hydroxyprogesterone or medroprogesterone), estrogens, (e.g.diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens (e.g.testosterone), aromatase inhibitors (e.g. aminogluthetimide),17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide,apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid,leuprolide (leuprorelin), luteinizing hormone-releasing hormone,pifithrin-α, rapamycin, sex hormone-binding globulin, and thapsigargin.

Exemplary therapeutics also include enzyme inhibitors such as,S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-dichlorobenz-imidazole1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin,2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin,trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

Exemplary therapeutics also include gene regulators such as5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D₃),4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal(vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoicacid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, andtroglitazone.

Exemplary therapeutics also include cytotoxic agents such as, forexample, the pteridine family of drugs, diynenes, and thepodophyllotoxins. Particularly useful members of those classes include,for example, methopterin, podophyllotoxin, or podophyllotoxinderivatives such as etoposide or etoposide phosphate, leurosidine,vindesine, leurosine and the like.

Still other cytotoxins that are compatible with the teachings hereininclude auristatins (e.g. auristatin E and monomethylauristan E),calicheamicin, gramicidin D, maytansanoids (e.g. maytansine),neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide,emetine, tenoposide, colchicin, dihydroxy anthracindione, mitoxantrone,procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs orhomologs thereof.

Other types of functional moieties are known in the art and can bereadily used in the methods and compositions of the present inventionbased on the teachings contained herein.

3.2. Chemistries for Linking Functional Moieties to Amino Acid SideChains

Chemistries for linking the foregoing functional moieties be they smallmolecules, nucleic acids, polymers, peptides, proteins,chemotherapeutics, or other types of molecules to particular amino acidside chains are known in the art (for a detailed review of specificlinkers see, for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press (1996)).

Exemplary art recognized linking groups for sulfhydryl moieties (e.g.,cysteine, or thiol side chain chemistries) include, but are not limitedto, activated acyl groups (e.g., alpha-haloacetates, chloroacetic acid,or chloroacetamide), activated alkyl groups, Michael acceptors such asmaleimide or acrylic groups, groups which react with sulfhydryl moietiesvia redox reactions, and activated di-sulfide groups. The sulfhydrylmoieties may also be linked by reaction with bromotrifluoroacetone,alpha-bromo-beta-(5-imidazoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

In a preferred embodiment, the cysteine or thiol side chain chemistry islinked during or subsequent to the production of an Fc containingpolypeptide. For example, when producing the modified Fc containingpolypeptide using cell culture, conditions are provided such that a freecysteine in solution can form a cysteine adduct with the thiol sidechain of the Fc containing polypeptide. The so formed adduct may be usedto inhibit glycosylation and/or effector function, or, subsequentlysubjected to reducing conditions to remove the adduct and thereby allowfor the use of one of the aforementioned sulfhydryl chemistries.

Exemplary art recognized linking groups for hydroxyl moieties (e.g.,serine, threonine, or tyrosine side chain chemistries) include thosedescribed above for sulfhydryl moieties including activated acyl groups,activated alkyl groups, and Michael acceptors.

Exemplary art recognized linking groups for amine moieties (e.g.,asparagine or arginine side chain chemistries) include, but are notlimited to, N-succinimidyl, N-sulfosuccinimidyl, N-phthalimidyl,N-sulfophthalimidyl, 2-nitrophenyl, 4-nitrophenyl, 2,4-dinitrophenyl,3-sulfonyl-4-nitrophenyl, 3-carboxy-4-nitrophenyl, imidoesters (e.g.,methyl picolinimidate), pyridoxal phosphate, pyridoxal,chloroborohydride, trinitrobenzenesulfonic acid, O-methyliosurea, and2,4-pentanedione.

Exemplary art recognized linking groups for acidic moieties (e.g.,aspartic acid or glutamic side chain chemistries) include activatedesters and activated carbonyls. Acidic moieties can also be selectivelymodified by reaction with carbodiimides (R′N—C—N—R′) such as1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

Where the functional moiety desired is a pegylation moiety, pegylationreactions known in the art are employed or as described herein (seealso, e.g., Example 3). For example, in one method, the pegylation iscarried out via an acylation reaction or an alkylation reaction with areactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer). A water-soluble polymer for pegylation of theantibodies and antibody fragments of the invention is polyethyleneglycol (PEG). In another embodiment, the polymer for pegylation ispolyethylene glycol-maleimide (i.e., PEG-maleimide).

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result. Inone embodiment, a particular amino acid reside can be targeted, forexample, the first amino acid residue altered in order to inhibitglycosylation of a second amino acid residue, and preferably where thefirst amino acid is a cysteine or has a thiol chemistry.

4. Expression of Recombinant Antibodies

The modified antibodies of the invention are typically produced byrecombinant expression. Nucleic acids encoding light and heavy chainvariable regions, optionally linked to constant regions, are insertedinto expression vectors. The light and heavy chains can be cloned in thesame or different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species.

Other microbes, such as yeast, are also useful for expression.Saccharomyces and Pichia are exemplary yeast hosts, with suitablevectors having expression control sequences (e.g., promoters), an originof replication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formethanol, maltose, and galactose utilization.

In addition to microorganisms, mammalian tissue culture may also be usedto express and produce the polypeptides of the present invention (e.g.,polynucleotides encoding immunoglobulins or fragments thereof). SeeWinnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various COS cell lines, HeLa cells, 293 cells, myeloma celllines, transformed B-cells, and hybridomas. Expression vectors for thesecells can include expression control sequences, such as an origin ofreplication, a promoter, and an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Coet al., J. Immunol. 148:1149 (1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

The antibodies of the invention can be expressed using a single vectoror two vectors. When the antibody heavy and light chains are cloned onseparate expression vectors, the vectors are co-transfected to obtainexpression and assembly of intact immunoglobulins. Once expressed, thewhole antibodies, their dimers, individual light and heavy chains, orother immunoglobulin forms of the present invention can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, HPLCpurification, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, N.Y., (1982)). Substantially pureimmunoglobulins of at least about 90 to 95% homogeneity are preferred,and 98 to 99% or more homogeneity most preferred, for pharmaceuticaluses.

5. Prophylactic, Diagnostic, and Therapeutic Methods

The present invention is also directed inter alia to the production ofaglycosylated antibodies suitable for the prognosis, diagnosis, ortreatment of diseases associated with immune disorders, including forexample, disorders where it is desirable to bind an antigen using atherapeutic antibody but refrain from triggering effector function.

Accordingly, in certain embodiments, the aglycosylated antibodies orantigen-binding fragments of the present invention are useful in theprevention or treatment of immune disorders including, for example,glomerulonephritis, scleroderma, cirrhosis, multiple sclerosis, lupusnephritis, atherosclerosis, inflammatory bowel diseases or rheumatoidarthritis. In another embodiment, the antibodies or antigen-bindingfragments of the invention can be used to treat or prevent inflammatorydisorders, including, but not limited to, Alzheimer's, severe asthma,atopic dermatitis, cachexia, CHF-ischemia, coronary restinosis, Crohn'sdisease, diabetic nephropathy, lymphoma, psoriasis,fibrosis/radiation-induced, juvenile arthritis, stroke, inflammation ofthe brain or central nervous system caused by trauma, and ulcerativecolitis.

Other inflammatory disorders which can be prevented or treated with theaglycosylated antibodies or antigen-binding fragments of the inventioninclude inflammation due to corneal transplantation, chronic obstructivepulmonary disease, hepatitis C, multiple myeloma, and osteoarthritis.

In another embodiment, the antibodies or Fc-containing fragments of theinvention can be used to prevent or treat neoplasia, including, but notlimited to bladder cancer, breast cancer, head and neck cancer, Kaposi'ssarcoma, melanoma, ovarian cancer, small cell lung cancer, stomachcancer, leukemia/lymphoma, and multiple myeloma. Additional neoplasiaconditions include, cervical cancer, colo-rectal cancer, endometrialcancer, kidney cancer, non-squamous cell lung cancer, and prostatecancer.

In another embodiment, the antibodies or antigen-binding fragments ofthe invention can be used to prevent or treat neurodegenerativedisorders, including, but not limited to Alzheimer's, stroke, andtraumatic brain or central nervous system injuries. Additionalneurodegenerative disorders include ALS/motor neuron disease, diabeticperipheral neuropathy, diabetic retinopathy, Huntington's disease,macular degeneration, and Parkinson's disease.

In still another embodiment, the antibody or Fc-containing fragment ofthe invention an be used to prevent or treat an infection caused by apathogen, for example, a virus, prokaryotic organism, or eukaryoticorganism.

In clinical applications, a subject is identified as having or at riskof developing one of the above-mentioned conditions by exhibiting atleast one sign or symptom of the disease or disorder. At least oneantibody or antigen-binding fragment thereof of the invention orcompositions comprising at least one antibody or antigen-bindingfragment thereof of the invention is administered in a sufficient amountto treat at least one symptom of a disease or disorder, for example, asmentioned above. In one embodiment, a subject is identified asexhibiting at least one sign or symptom of a disease or disorderassociated with detrimental CD154 activity (also known as CD40 ligand orCD40L; see, e.g., Yamada et al., Transplantation, 73:S36-9 (2002);Schonbeck et al., Cell. Mol. Life Sci. 58:4-43 (2001); Kirk et al.,Philos. Trans. R. Soc. Lond. B. Sci. 356:691-702 (2001); Fiumara et al.,Br. J. Haematol. 113:265-74 (2001); and Biancone et al., Int. J. Mol.Med. 3(4):343-53 (1999)).

Accordingly, an aglycosylated antibody of the invention is suitable foradministration as a therapeutic immunological reagent to a subject underconditions that generate a beneficial therapeutic response in a subject,for example, for the prevention or treatment of a disease or disorder,as for example, described herein.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, for example as described herein, homogeneouspeptides of at least 99% w/w can be obtained.

The methods can be used on both asymptomatic subjects and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a subject by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the subject. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thesubject. In exemplary embodiments, the subject is monitored for thelevel of administered antibody in the blood of the subject.

The invention thus fulfills a longstanding need for therapeutic regimesfor preventing or ameliorating immune conditions, for example,CD154-associated immune conditions.

It is also understood the antibodies of the invention are suitable fordiagnostic or research applications, especially, for example, andiagnostic or research application comprising a cell-based assay wherereduced effector function is desirable.

6. Animal Models for Testing the Efficacy of Aglycosylated Antibodies

An antibody of the invention can be administered to a non-human mammalin need of, for example, an aglycosylated antibody therapy, either forveterinary purposes or as an animal model of human disease, e.g., animmune disease or condition stated above. Regarding the latter, suchanimal models may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of effector function,dosages, and time courses of administration).

Examples of animal models which can be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating rheumatoid arthritis (RA) includeadjuvant-induced RA, collagen-induced RA, and collagen mAb-induced RA(Holmdahl et al., (2001) Immunol. Rev. 184:184; Holmdahl et al., (2002)Ageing Res. Rev. 1:135; Van den Berg (2002) Curr. Rheumatol. Rep.4:232).

Examples of animal models which can be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating inflammatory bowel disease (IBD)include TNBS-induced IBD, DSS-induced IBD, and (Padol et al. (2000) Eur.J. Gastrolenterol. Hepatol. 12:257; Murthy et al. (1993) Dig. Dis. Sci.38:1722).

Examples of animal models which can be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating glomerulonephritis includeanti-GBM-induced glomerulonephritis (Wada et al. (1996) Kidney Int.49:761-767) and anti-thy 1-induced glomerulonephritis (Schneider et al.(1999) Kidney Int 56:135-144).

Examples of animal models which can be used for evaluating thetherapeutic efficacy of antibodies or antigen-binding fragments of theinvention for preventing or treating multiple sclerosis includeexperimental autoimmune encephalomyelitis (EAE) (Link and Xiao (2001)Immunol. Rev. 184:117-128).

Animal models can also be used for evaluating the therapeutic efficacyof antibodies or antigen-binding fragments of the invention forpreventing or treating CD154-related conditions, such as systemicerythematosus lupus (SLE), for example using the MRL-Fas^(lpr) mice(Schneider, supra; Tesch et al. (1999) J. Exp. Med. 190).

7. Treatment Regimes and Dosages

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a subject suffering from a disorder treatable with apolypeptide having an Fc region, for example, an immune system disorder,in an amount sufficient to eliminate or reduce the risk, lessen theseverity, or delay the outset of the disorder, including biochemical,histologic and/or behavioral symptoms of the disorder, its complicationsand intermediate pathological phenotypes presenting during developmentof the disorder. In therapeutic applications, compositions ormedicaments are administered to a subject suspected of, or alreadysuffering from such a disorder in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disorder (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disorder. Thepolypeptides of the invention are particularly useful for modulating thebiological activity of a cell surface antigen that resides in the blood,where the disease being treated or prevented is caused at least in partby abnormally high or low biological activity of the antigen.

In some methods, administration of agent reduces or eliminates theimmune disorder, for example, inflammation, such as associated withCD154 activity. An amount adequate to accomplish therapeutic orprophylactic treatment is defined as a therapeutically- orprophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the subject, whether the subject is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the subject is a human butnon-human mammals including transgenic mammals can also be treated.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 20 mg/kg, of the host bodyweight. For example dosages can be 1 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. In somemethods, dosage is adjusted to achieve a plasma antibody concentrationof 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively,antibody can be administered as a sustained release formulation, inwhich case less frequent administration is required. Dosage andfrequency vary depending on the half-life of the antibody in thesubject. In general, human antibodies show the longest half-life,followed by humanized antibodies, chimeric antibodies, and nonhumanantibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a subject not already in thedisease state to enhance the subject's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the subject's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somesubjects continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the subject shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per subject. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof a protein drug is intravascular, subcutaneous, or intramuscular,although other routes can be effective. In some methods, agents areinjected directly into a particular tissue where deposits haveaccumulated, for example intracranial injection. In some methods,antibodies are administered as a sustained release composition ordevice, such as a Medipad™ device. The protein drug can also beadministered via the respiratory tract, e.g., using a dry powderinhalation device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofimmune disorders.

8. Pharmaceutical Compositions

The therapeutic compositions of the invention include at least oneaglycosylated antibody or antibody fragment of the invention in apharmaceutically acceptable carrier. A “pharmaceutically acceptablecarrier” refers to at least one component of a pharmaceuticalpreparation that is normally used for administration of activeingredients. As such, a carrier may contain any pharmaceutical excipientused in the art and any form of vehicle for administration. Thecompositions may be, for example, injectable solutions, aqueoussuspensions or solutions, non-aqueous suspensions or solutions, solidand liquid oral formulations, salves, gels, ointments, intradermalpatches, creams, lotions, tablets, capsules, sustained releaseformulations, and the like. Additional excipients may include, forexample, colorants, taste-masking agents, solubility aids, suspensionagents, compressing agents, enteric coatings, sustained release aids,and the like.

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)).

9. Monitoring the Course of Treatment

Treatment of a subject suffering from a disease or disorder, such as animmune disorder, can be monitored using standard methods. Some methodsentail determining a baseline value, for example, of an antibody levelor profile in a subject, before administering a dosage of agent, andcomparing this with a value for the profile or level after treatment. Asignificant increase (i.e., greater than the typical margin ofexperimental error in repeat measurements of the same sample, expressedas one standard deviation from the mean of such measurements) in valueof the level or profile signals a positive treatment outcome (i.e., thatadministration of the agent has achieved a desired response). If thevalue for immune response does not change significantly, or decreases, anegative treatment outcome is indicated.

In other methods, a control value (i.e., a mean and standard deviation)of level or profile is determined for a control population. Typicallythe individuals in the control population have not received priortreatment. Measured values of the level or profile in a subject afteradministering a therapeutic agent are then compared with the controlvalue. A significant increase relative to the control value (e.g.,greater than one standard deviation from the mean) signals a positive orsufficient treatment outcome. A lack of significant increase or adecrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other methods, a control value of the level or profile (e.g., a meanand standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a subject are compared with thecontrol value. If the measured level in a subject is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a subject issignificantly below the control value, continued administration of agentis warranted. If the level in the subject persists below the controlvalue, then a change in treatment may be indicated.

In other methods, a subject who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodylevels or profiles to determine whether a resumption of treatment isrequired. The measured level or profile in the subject can be comparedwith a value previously achieved in the subject after a previous courseof treatment. A significant decrease relative to the previousmeasurement (i.e., greater than a typical margin of error in repeatmeasurements of the same sample) is an indication that treatment can beresumed. Alternatively, the value measured in a subject can be comparedwith a control value (mean plus standard deviation) determined in apopulation of subjects after undergoing a course of treatment.Alternatively, the measured value in a subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a subject.

The antibody profile following administration typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered. For example the half-life of some humanantibodies is of the order of 20 days.

In some methods, a baseline measurement of antibody to a given antigenin the subject is made before administration, a second measurement ismade soon thereafter to determine the peak antibody level, and one ormore further measurements are made at intervals to monitor decay ofantibody levels. When the level of antibody has declined to baseline ora predetermined percentage of the peak less baseline (e.g., 50%, 25% or10%), administration of a further dosage of antibody is administered. Insome methods, peak or subsequent measured levels less background arecompared with reference levels previously determined to constitute abeneficial prophylactic or therapeutic treatment regime in othersubjects. If the measured antibody level is significantly less than areference level (e.g., less than the mean minus one standard deviationof the reference value in population of subjects benefiting fromtreatment) administration of an additional dosage of antibody isindicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom (e.g., physical or mental symptom)routinely relied on by researchers or physicians to diagnose or monitordisorders.

The following examples are included for purposes of illustration andshould not be construed as limiting the invention.

EXEMPLIFICATION

Throughout the examples, the following materials and methods were usedunless otherwise stated.

Materials and Methods

In general, the practice of the present invention employs, unlessotherwise indicated, conventional techniques of chemistry, molecularbiology, recombinant DNA technology, immunology (especially, e.g.,antibody technology), and standard techniques in electrophoresis. See,e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold SpringHarbor Laboratory Press (1989); Antibody Engineering Protocols (Methodsin Molecular Biology), 510, Paul, S., Humana Pr (1996); AntibodyEngineering: A Practical Approach (Practical Approach Series, 169),McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlowet al., C.S.H.L. Press, Pub. (1999); and Current Protocols in MolecularBiology, eds. Ausubel et al., John Wiley & Sons (1992).

Production of the Modified Antibodies

For producing the modified antibodies of the invention, polynucleotidesencoding either a model human antibody (hu5c8), variant antibodiesthereof, or corresponding Fc regions, were introduced into standardexpression vectors. The human antibody hu5c8 and variants thereof aredescribed in, e.g., U.S. Pat. Nos. 5,474,771 and 6,331,615. The cDNAsequence and amino acid sequence are provided in the sequence listingfor, respectively, the hu5c8 IgG1 heavy chain (SEQ ID NOS: 1-2), hu5c8light chain (SEQ ID NOS: 3-4), hu5c8 IgG1 Fc region (SEQ ID NOS: 5-6),hu5C8 IgG4 heavy chain (SEQ ID NOS: 7-8), hu5c8 IgG4 variant (S228P)(SEQ ID NOS: 9-10), and hu5c8 IgG4 variant (S228P/T299A) (SEQ ID NOS:11-12). Vectors where then introduced into EBNA 293 cells usinglarge-scale transient transfection techniques. The transfected 293 cellswere cultured using standard media and incubation conditions. Cells weretypically refed after 1 day post-transfection and then allowed toexpress and secrete the recombinant protein for 1 to 3 days. Culturemedia containing the secreted recombinant antibodies or Fc regions werethen harvested for purification.

Purification of the Modified Antibodies

For performing antibody purification, recombinant aglycosylatedantibodies produced in eukaryotic cells were harvested from the cellculture medium and subjected to the following chromatography techniques.In particular, recombinant Protein A columns (5 mL) were prepared andwashed with 100 mL 0.1 N NaOH and then equilibrated with PBS untilneutralized. The conditioned media (˜1.5 L) was then pumped through thecolumn at 10 mL/min. After loading, the column was washed with 100 mL3×PBS and then 10 mL 1×PBS. The antibodies were eluted with 1.3 mLfractions of 100 mM NaH₂PO₄, pH 2.8 into collection tubes containing 0.3mL 1 M HEPES, pH 8 for immediate neutralization. Fractions containingthe eluted antibodies were identified by monitoring the concentrationusing light absorbance (A280) of 1:10 dilutions of each fraction. Thispurification step was scaled up or down proportionately to the scale ofthe transient transfection.

Resultant Protein A pools were further purified by chromatography on a1.6 mL Poros HS column. The recombinant protein pools (˜8 mL) werediluted ten-fold with 25 mM NaAcetate, pH 4.5 and half was loaded ineach of two purification runs using a BioCad HPLC. The proteins wereloaded at a flow rate of 5 mL/min, the column washed with 10 columnvolumes of the dilution buffer and then eluted with a 25 column volumegradient of 0 to 1 M NaCl in the dilution buffer. Fractions of 0.8 mLwere collected and monitored for protein concentration by lightabsorbance (A280).

Alternatively, the resultant Protein A pool from a small scalepreparation was purified by Protein L chromatography. A Protein L column(1 mL) was prepared and washed with 10 mL 0.1 N NaOH and thenequilibrated with PBS until neutralized. The neutralized Protein A pool(3 mL) was then loaded in 1 mL aliquots. After loading, the column waswashed with 10 mL 3×PBS and then 10 mL 1×PBS. The antibodies were elutedwith 0.4 mL fractions of 100 mM NaH₂PO₄, pH 2.8 into collection tubescontaining 0.1 mL 1 M HEPES, pH 8 for immediate neutralization.Fractions containing the eluted antibodies were identified by monitoringthe concentration using light absorbance (A280) of 1:5 dilutions of eachfraction.

In addition to light absorbance, eluants containing recombinant proteinwere also monitored with a refractive index detector (Waters) and aPrecision Detector PD2020 light scattering instrument. Molecular weightswere calculated with the Precision Detector software. All variantantibodies (four forms of hu5c8) eluted identically from the SEC column,showing a single major peak with a minor amount of higher molecularweight material (dimer). A molecular weight of 148,300 was determined bylight scattering for the main peak of the T299C hu5c8 variant. Sizeexclusion chromatography of the huIgG1 Fc variants was carried outidentically to the full length antibodies. All four Fc proteins ranidentically, giving a major peak with calculated MWs ranging from 53,000to 55,000 Daltons. Finally, recombinant protein samples were obtained,dialyzed against PBS, sterile filtered, and stored at 4° C. in 10 mgaliquots until needed for further analysis.

SDS-PAGE

For performing SDS-PAGE, protein samples were typically diluted to 200μg/mL in Laemmli SDS-PAGE sample buffer containing either 25 mM DTT forreducing conditions, or 25 mM NEM for non-reducing conditions. Aliquotsof 2.5 and 10 μl were loaded on 4-20% gradient gels.

Mass Spectrometry

For performing mass spectroscopy, protein samples were reduced in 9 mMDTT, at pH 7.8, prior to analysis. The samples were desalted over a C4guard column and analyzed on-line by ESMS using a triple quadrupoleinstrument. The ESMS raw data were deconvoluted by the MaxEnt program togenerate zero charged mass spectra. This procedure allows for multiplecharged signals to collapse into one peak for molecular massdeterminations.

Pegylation

For performing pegylation of the aglycosylated polypeptides of theinvention, aliquots of 50 μL of 0.94 mg/mL solutions of the T299A andT299C variant Fc were first precipitated with 1 mL ethanol at −20° C.overnight. Resultant precipitates were then pelleted and the ethanolremoved and 50 μL of a solution of 6.4 M urea, 2% SDS and 10 mM EDTA, pH8 was added and the solution heated to 100° C. for 5 min. For reduction,half the samples were treated with 4 mM TCEP for 30 min at roomtemperature. Aliquots of 5 μL of 1 M MES buffer at pH 6.5 were thenadded followed by either 50 μL H2O or a 5 mM solution of PEG(5K)-maleimide. After 30 min at room temperature, 10 μL aliquots of a 4×solution of Laemmli SDS-PAGE sample buffer was added to 30 μL of thereaction mixtures and the solution heated to 100° C. for 5 min. Then 5and 15 μL aliquots of recombinant protein were loaded on 4-20% gradientgels for a determination of relative amounts of pegylation thatoccurred.

Example 1 Methods for Producing and Characterizing AglycosylatedAntibodies

The following example describes the production of an aglycosylatedantibody in a eukaryotic cell and the characterization of the resultantantibody.

Nucleic acids encoding a model human antibody (hu5c8) of the IgG1subtype having binding affinity for the CD154 ligand were geneticallyengineered to have one of several alterations. The first alterationcomprised a codon encoding in place of the wild type amino acid residue,i.e., threonine, at position 299, an alanine (T299A). In anotheralteration, the codon encoding threonine at position 299 was changed toencode a cysteine (T299A). A control alteration was also included, inwhich the specific asparagine that is glycosylated is mutated (N297Q)(FIGS. 3, 5-7). In addition, the T299A mutation was introduced into amodel human antibody hu5C8 of the IgG4 subtype. The IgG4 sequence had afurther modification in the hinge peptide (S228P) to stabilize theinterchain disulfides, an issue unrelated to the aglycosyl modification(FIG. 4). Each alteration was incorporated into an expression vector andintroduced into a eukaryotic cell line using the methods describedherein. In addition, the forgoing alterations where also tested in thecontext of an Fc region unlinked from the corresponding variable region.Each modified antibody, or Fc fragment thereof, along with acorresponding control antibody or antibody fragment, was then expressedin cell culture, harvested from the cell culture media, and purifiedusing standard techniques. Each antibody or antibody fragment was thencharacterized for its aglycosylation and binding activity.

The aglycosylation for each antibody or antibody fragment wascharacterized using standard gel electrophoresis and chromatographytechniques. In particular, reducing and non-reducing SDS-PAGE and sizeexclusion chromatography under native conditions were performed anddemonstrated that the T299A and T299C variants of test antibody (hu5c8)and fragments thereof, i.e., huIgG1 Fc, were of the expected molecularsize and subunit organization. The absence of glycosylation of the T299Aand T299C antibody variants was indicated by the more rapid migration ofthe heavy chain of the proteins on reducing SDS-PAGE (FIG. 3). Inaddition, mass spectrometry under reducing conditions confirmed theexpected mass of the constructs and the absence of glycans in the T299Aand T299C variants (FIGS. 8-11). Mass spectroscopy under non-reducingconditions also demonstrated the presence of cysteine adducts on thehuIgG1 T299C Fc variants (FIGS. 8-11).

The mass of the T299A variant corresponded to the predicted proteindimer (expected, 51,824.7, found, 51,826). In contrast the mass of theT299C variant was 246 Daltons larger that predicted (expected 51,886,found 52,132) (FIG. 3). This would correspond to the addition of twocysteine adducts to the Fc dimer (2×120=240) (FIG. 5).

Accordingly, it was concluded that the alteration of the first aminoacid proximal to a glycosylation motif inhibited the glycosylation ofthe antibody at second amino acid residue thereby providing an efficientand reliable approach for producing aglycosylated antibodies ineukaryotic cells.

Example 2 Methods for Producing and Aglycosylated Antibody with ReducedEffector Function using Amino Acid Substitutions of Sufficient StericBulk and/or Charge

The following example describes the production of an aglycosylatedantibody by altering an antibody at a first amino acid residue with aresidue that has sufficient steric bulk and/or charge as to inhibitglycosylation.

Nucleic acids encoding a candidate antibody, for example, an antibody ofthe IgG1 or IgG4 subtype, were genetically engineered to have one ofseveral alterations predicted to inhibit glycosylation and/or effectorfunction. While not wishing to be bound by theory, results obtainedabove for a cysteine adduct support the rationale that a sufficientlybulky and/or charged residue will inhibit a glycosidase fromglycosylating an Fc-containing polypeptide and reduce undesired effectorfunction. For example, a substitution at the Kabat position of 299(e.g., T299) with a bulky or charged residue is predicted to inhibit aglycosidase from glycosylating the antibody at, for example, position297. In addition, such an amino acid substitution is also predicted tomodulate the binding of the antibody to an Fc receptor. In the boundcomplex between an antibody Fc region and an Fc receptor, for example,the FcγIIIb receptor, the residue T299 of the antibody Fc region islocated very close to the binding interface with the FcγIIIb receptor.In particular, the distances of the side chain chemistry of the T299residue to the Y150 and H152 residues of the FcγIIIb receptor are 4.2 Åand 5.6 Å, respectively. Thus, by substituting T299 for a residue withsufficient steric bulk, such as F, H, Q, W, or Y, the antibody will notonly be aglycosylated but also have reduced Fc binding affinity to theFc receptor due to unfavorable steric interactions.

Still further, the inhibition of glycosylation and Fc binding can bemodulated by substituting T299 with a charged side chain chemistry suchas D, E, K, or R. The resulting antibody variant will not only havereduced glycosylation but also reduced Fc binding affinity to an Fcreceptor due to unfavorable electrostatic interactions.

Accordingly, modifying a first amino acid residue side chain chemistryto one of sufficient steric bulk and/or charge, is predicted to inhibitthe glycosylation of the antibody at a second amino acid residue as wellas reduce Fc binding to an Fc receptor. Thus, the invention provides anefficient and reliable approach for producing aglycosylated antibodieswith reduced effector function in eukaryotic cells.

Example 3 Methods for Pegylating Aglycosylated Antibodies

The following example describes the production of an aglycosylatedantibody in a eukaryotic cell and the pegylation of the resultantantibody.

In particular, the T299C antibody variant was determined to bespecifically modified with Peg-maleimide under non-denaturing conditionsby first reducing the protein with TCEP to remove the cysteine adduct,allowing the hinge disulfides to reform by dialyzing the protein overseveral days, and reacting with PEG-maleimide. The T299A antibodyvariant could not be modified with PEG under these conditions (FIG. 6).

Briefly, to reduce the test proteins, 200 μL of the 0.94 mg/mL T299A andT299C Fc antibody variant preparations were treated with 4 μL of 500 mMEDTA, pH 8 (final concentration 10 mM) and 10 μL of 100 mM TCEP (finalconcentration 5 mM) for 3 hours at room temperature. The reducedproteins were dialyzed against PBS over four days with five changes at1:1000 volume ratios. Aliquots (5 μL) of the protein preparations werethen treated with 5 μL of 5 mM PEG-maleimide (5,000 mw) undernon-denaturing conditions for 1 h and then prepared for SDS-PAGE by theaddition of 5 μL of 4× Laemmli SDS-PAGE sample buffer contained 100 mMDTT. Only the T299C antibody variant was observed to have a PEG adduct(FIG. 7).

Corroboration that the T299C cysteine had formed a cystine disulfidebond was obtained by attempting to react the Fc with the thiol-specificmodifying reagent, PEG-maleimide. Under denaturing (6.4 M urea, 2% SDS),but non-reducing conditions, no reaction occurred with thePEG-maleimide. Under reducing conditions the T299C variant did reactwith the PEG-maleimide, yielding a larger product than the T299Avariant, indicating the presence of the extra cysteine (FIG. 3).

Accordingly, it was concluded that the alteration of the first aminoacid proximal to a glycosylation motif capable of inhibiting theglycosylation of the antibody at a second amino acid residue, whenaltered to a cysteine residue, also provided for an efficient andreliable pegylation residue.

Example 4 Methods for Determining Altered Effector Function ofAglycosylated Antibodies

The following example describes assays for determining the alteredeffector function of the aglycosylated antibodies of the invention.

The effector function of the aglycosylated variant antibodies of theinvention were characterized by their ability to bind an antigen andalso bind an Fc receptor or a complement molecule such as C1q. Inparticular, the FcγR binding affinities were measured with assays basedon the ability of the antibody to form a “bridge” between the CD154antigen and a cell bearing an Fc receptor. The C1q binding affinity wasmeasured based on the ability of the antibody to form a “bridge” betweenthe CD154 antigen and C1q (FIGS. 14-15).

Briefly, the FcγR bridging assay was performed by coating 96 wellMaxisorb ELISA plates (Nalge-Nunc Rochester, N.Y., USA) with recombinantsoluble human CD154 ligand (i.e., at a concentration of 1 μg/mlovernight at 4° C. in PBS; Karpusas, Hsu et al. 1995). Titrations ofglycosylated or aglycosylated forms of anti-CD154 antibody (hu5c8) werethen bound to CD154 for 30 minutes at 37° C., the plates were thenwashed, and the binding of fluorescently labeled U937 (CD64⁺) cells wasmeasured. The U937 cells were grown in RPMI medium with 10% FBS, 10 mMHEPES, L-glutamine, and penicillin/streptomycin, split 1:2, andactivated for one day prior to the assay with 1000 units/ml of IFNγ toincrease Fc receptor (FcγRI) expression.

In another variation of the assay, the ability of the antibodies of theinvention to bind to, or rather, fail to bind, to yet another Fcreceptor, in particular, FcγRIII (CD16) was performed using the abovebridging assay against fluorescently labeled human T cells (Jurkatcells) transfected with a CD16 expression construct. The ligand wasproduced by a monolayer of CD154-expressing Chinese Hamster Ovary (CHO)cells grown in 96 well tissue culture plates (Corning Life SciencesActon, Mass., USA). The CHO-CD154⁺ cells were seeded into 96 well platesat 1×10⁵ cells/ml and grown to confluency in α⁻MEM with 10% dialyzedFBS, 100 nM methotrexate, L-glutamine, and penicillin/streptomycin(Gibco-BRL Rockville, Md., USA). The CD16⁺ Jurkat cells were grown inRPMI with 10% FBS, 400 μg/ml Geneticin, 10 mM HEPES, sodium pyruvate,L-glutamine, and penicillin/streptomycin (Gibco-BRL) and split 1:2 oneday prior to performing the assay.

In the assays for both receptors, the Fc receptor-bearing cells werelabeled with 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Molecular Probes Eugene, Oreg., USA) for20 minutes at 37° C. After washing to remove excess label, 1×10⁵ of thelabeled cells were incubated in the assay for 30 minutes at 37° C.Unbound FcγR positive cells were removed by washing several times andplates were read on a microplate reader (Cytofluor 2350 FluorescentMicroplate Reader, Millipore Corporation Bedford, Mass., USA) at anexcitation wavelength of 485 nm and an emission wavelength of 530 nm.

In each bridging assay, a reduced effector function of the aglycosylatedIgG1 antibody variants of the invention as a function of FcγRI (upperpanel) or FcγRIII (lower) binding was observed (FIGS. 12-13). Inparticular, the T299C variant, which is both aglycosylated and capableof forming a cysteine adduct was observed to have less effector function(FcγRI binding) as compared to merely aglycosylated antibodies (FIG. 12upper panel). The aglycosyl IgG4 T299A antibody variant was also foundto have exceptionally low binding to FcRγI, lower than the IgG1 T299Avariant. This was not expected since the glycosylated IgG1 and IgG4antibodies show similar binding in this assay (FIG. 13).

The C1q binding assay was performed by coating 96 well Maxisorb ELISAplates (Nalge-Nunc Rochester, N.Y., USA) with 50 μl recombinant solublehuman CD154 ligand (Karpusas et al. Structure, 15; 3(12):1426 (1995) at10 μg/ml overnight at 4° C. in PBS. The wells were aspirated and washedthree times with wash buffer (PBS, 0.05% Tween 20) and blocked for ≧1 hwith 200 μl/well of block/diluent buffer (0.1 M Na₂HPO₄, pH 7, 0.1 MNaCl, 0.05% Tween 20, 0.1% gelatin). The antibody to be tested wasdiluted in block/diluent buffer starting at 15 μg/ml with 3-folddilutions. 50 μl were added per well, and the plates incubated for 2 hat room temperature. After aspirating and washing as above, 50 μl/wellof 2 μg/ml of Sigma human C1q (C0660) diluted in block/diluent bufferwas added and incubated for 1.5 h at room temperature. After aspiratingand washing as above, 50 μl/well of sheep anti C1q (Serotec AHP033),diluted 3,560-fold in block/diluent buffer, was added. After incubationfor 1 h at room temperature, the wells were aspirated and washed asabove. 50 μl/well of donkey anti-sheep IgG HRP conjugate (JacksonImmunoResearch 713-035-147) diluted to 1:10,000 in block/diluent wasthen added, and the wells incubated for 1 h at room temperature. Afteraspirating and washing as above, 100 μl TMB substrate (420 μM TMB,0.004% H₂O₂ in 0.1 M sodium acetate/citric acid buffer, pH 4.9) wasadded and incubated for 2 min before the reaction was stopped with 100μl 2 N sulfuric acid. The absorbance was read at 450 nm with a SoftmaxPRO instrument, and Softmax software was used to determine the relativebinding affinity (C value) with a 4-parameter fit.

As shown in FIGS. 14-15, the T299C mutant had a C1q binding affinitythat was not only below the hu5c8 antibody but below that of theaglycosylated N297Q and T299A variants, which indicates that themutation to cysteine was unexpectedly beneficial. The IgG4 T299A mutantshowed no binding to C1q, similarly to the aglycosylated IgG4.

Accordingly, it was concluded that the alteration of a first amino acidproximal to a glycosylation motif inhibited the glycosylation of theantibody at a second amino acid residue, and when the first amino acidwas a cysteine residue, the antibody had more reduced effector function.In addition, inhibition of glycosylation of an antibody of the IgG4subtype had a more profound affect on FcγRI binding than expected.

EQUIVALENTS

For one skilled in the art, using no more than routine experimentation,there are many equivalents to the specific embodiments of the inventiondescribed herein. Such equivalents are intended to be encompassed by thefollowing claims.

1. A nucleic acid molecule encoding a variant polypeptide of a parentpolypeptide comprising an Fc region, wherein the Fc region of thevariant polypeptide comprises a modified first amino acid residue atT299 of the Fc region according to the Kabat numbering system of IgGimmunoglobulins, the modified first amino acid residue having a sidechain chemistry selected from the group consisting of a side chainchemistry comprising a cysteine thiol, a side chain chemistry ofsufficient steric bulk such that the polypeptide displays reducedeffector function, and a side chain chemistry of sufficientelectrostatic charge such that the polypeptide displays reduced effectorfunction, and a second amino acid residue at N297 of the Fc regionaccording to the Kabat numbering system of IgG immunoglobulins, thesecond amino acid having reduced glycosylation, wherein the variantpolypeptide has reduced effector function as compared to the parentpolypeptide.
 2. The nucleic acid molecule of claim 1, wherein the sidechain chemistry of the modified first amino acid residue comprises acysteine thiol.
 3. The nucleic acid molecule of claim 1, wherein theside chain chemistry is of sufficient steric bulk such that thepolypeptide displays reduced effector function.
 4. The nucleic acidmolecule of claim 3, wherein the side chain chemistry of sufficientsteric bulk is that of an amino acid residue selected from the groupconsisting of Phe, Trp, His, Glu, Gln, Arg, Lys, Met, and Tyr.
 5. Thenucleic acid molecule of claim 1, wherein the side chain chemistry is ofsufficient electrostatic charge such that the variant polypeptidedisplays reduced effector function.
 6. The nucleic acid molecule ofclaim 5, wherein the side chain chemistry is that of an amino acidresidue selected from the group consisting of Asp, Glu, Lys, Arg, andHis.
 7. The nucleic acid molecule of claim 1, wherein the first aminoacid residue is modified by substitution with a replacement amino acid.8. The nucleic acid molecule of claim 1, wherein the polypeptide is anantibody variant.
 9. The nucleic acid molecule of claim 1, wherein theFc region is obtained from an antibody of an isotype selected from thegroup consisting of IgG1, IgG2, IgG3, and IgG4.
 10. A nucleic acidmolecule encoding a variant polypeptide of a parent polypeptidecomprising an IgG1 Fc region, wherein the Fc region of the variantpolypeptide comprises an amino acid at position 299 which differs fromthe amino acid at position 299 of the parent polypeptide, wherein theamino acid at position 299 of the parent polypeptide is threonine andthe amino acid at position 299 of the variant polypeptide is selectedfrom the group consisting of alanine, asparagine, glycine, tyrosine,cysteine, histidine, glutamic acid, aspartic acid, lysine, arginine,isolucine, leucine, methionine, phenylalanine, proline, tryptophan, andvaline, and wherein the variant polypeptide comprises an amino acid atposition 297 of the Fc region having reduced glycosylation and displaysreduced effector function as compared to the parent polypeptide.
 11. Thenucleic acid molecule of claim 10, wherein the reduced effector functionis reduced binding to an Fc receptor (FcR).
 12. The nucleic acidmolecule of claim 11, wherein the binding is reduced by a factorselected from the group consisting of about 1-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about8-fold, about 9-fold, about 10-fold, about 15-fold, about 50-fold, andabout 100-fold.
 13. The nucleic acid molecule of claim 10, wherein theFc receptor (FcR) is selected from the group consisting of FcγRI,FcγRII, and FcγRIII.
 14. The nucleic acid molecule of 10, wherein thereduced effector function is reduced binding to a complement protein.15. The nucleic acid molecule of claim 10, wherein the amino acid atposition 299 is a cysteine.
 16. The nucleic acid molecule of claim 10,wherein the amino acid at position 299 is an alanine.
 17. The nucleicacid molecule of claim 10, wherein the amino acid at position 299 isasparagine.
 18. The nucleic acid molecule of claim 10, wherein the aminoacid at position 299 is glycine.
 19. The nucleic acid molecule of claim10, wherein the amino acid at position 299 is tyrosine.
 20. The nucleicacid molecule of claim 10, wherein the amino acid at position 299 ishistidine.
 21. The nucleic acid molecule of claim 10, wherein the aminoacid at position 299 is glutamic acid.
 22. The nucleic acid molecule ofclaim 10, wherein the amino acid at position 299 is aspartic acid. 23.The nucleic acid molecule of claim 10, wherein the amino acid atposition 299 is a lysine.
 24. The nucleic acid molecule of claim 10,wherein the amino acid at position 299 is an arginine.
 25. The nucleicacid molecule of claim 10, wherein the amino acid at position 299 isisoleucine.
 26. The nucleic acid molecule of claim 10, wherein the aminoacid at position 299 is leucine.
 27. The nucleic acid molecule of claim10, wherein the amino acid at position 299 is methionine.
 28. Thenucleic acid molecule of claim 10, wherein the amino acid at position299 is phenylalanine.
 29. The nucleic acid molecule of claim 10, whereinthe amino acid at position 299 is proline.
 30. The nucleic acid moleculeof claim 10, wherein the amino acid at position 299 is tryptophan. 31.The nucleic acid molecule of claim 10, wherein the amino acid atposition 299 is valine.
 32. A nucleic acid molecule encoding a antibodyvariant of a parent antibody, wherein the antibody variant comprises anantigen-binding variable region and an Fc region comprising areplacement amino acid substituted for the threonine at position 299 ofthe Fc region according to the Kabat numbering system for IgGimmunoglobulins, the substituted amino acid residue having a side chainchemistry selected from the group consisting of a side chain chemistrycomprising a cysteine thiol, a side chain chemistry of sufficient stericbulk such that the polypeptide displays reduced effector function, and aside chain chemistry of sufficient electrostatic charge such that thepolypeptide displays reduced effector function and a second amino acidresidue at N297 of the Fc region according to the Kabat numbering systemfor IgG immunoglobulins, the second amino acid residue having reducedglycosylation as compared to the parent antibody.
 33. The nucleic acidmolecule of claim 32, wherein the Fc region is obtained from an antibodyof an isotype selected from the group consisting of IgG1, IgG2, IgG3,and IgG4.
 34. The nucleic acid molecule of claim 32, wherein the aminoacid at position 299 is asparagine.
 35. The nucleic acid molecule ofclaim 32, wherein the amino acid at position 299 is glycine.
 36. Thenucleic acid molecule of claim 32, wherein the amino acid at position299 is tyrosine.
 37. The nucleic acid molecule of claim 32, wherein theamino acid at position 299 is histidine.
 38. The nucleic acid moleculeof claim 32, wherein the amino acid at position 299 is glutamic acid.39. The nucleic acid molecule of claim 32, wherein the amino acid atposition 299 is aspartic acid.
 40. The nucleic acid molecule of claim32, wherein the amino acid at position 299 is lysine.
 41. The nucleicacid molecule of claim 32, wherein the amino acid at position 299 isarginine.
 42. The nucleic acid molecule of claim 32, wherein the aminoacid at position 299 is isoleucine.
 43. The nucleic acid molecule ofclaim 32, wherein the amino acid at position 299 is leucine.
 44. Thenucleic acid molecule of claim 32, wherein the amino acid at position299 is methionine.
 45. The nucleic acid molecule of claim 32, whereinthe amino acid at position 299 is phenylalanine.
 46. The nucleic acidmolecule of claim 32, the amino acid at position 299 is proline.
 47. Thenucleic acid molecule of claim 32, wherein the amino acid at position299 is tryptophan.
 48. The nucleic acid molecule of claim 32, whereinthe amino acid at position 299 is valine.
 49. The nucleic acid moleculeof claim 32, wherein the Fc region is from an IgG1 antibody.
 50. Thenucleic acid molecule of claim 32, wherein the Fc region is from an IgG4antibody.
 51. The nucleic acid molecule of claim 50, wherein thereplacement amino acid at position 299 is a cysteine.
 52. The nucleicacid molecule of claim 50, wherein the Fc region further comprises anamino acid at position 228 which differs from the amino acid at position228 of the parent antibody, wherein the amino acid at position 228 ofthe parent antibody is a serine and the amino acid at position 228 ofthe variant antibody is a proline.
 53. The nucleic acid molecule ofclaim 50, wherein the amino acid at position 299 is alanine.
 54. Anucleic acid molecule encoding a variant antibody of a parent antibodycomprising an IgG1 Fc region, wherein the Fc region of the variantantibody comprises an amino acid at position 299 which differs from theamino acid at position 299 of the parent antibody, wherein the aminoacid at position 299 of the parent antibody is threonine and the aminoacid at position 299 of the variant antibody is selected from the groupconsisting of alanine, asparagine, glycine, tyrosine, cysteine,histidine, glutamic acid, aspartic acid, lysine, arginine, isolucine,leucine, methionine, phenylalanine, proline, tryptophan, and valine, andwherein the variant antibody comprises an amino acid at position 297 ofthe Fc region having reduced glycosylation and displays reduced effectorfunction as compared to the parent polypeptide.
 55. The nucleic acidmolecule of claim 54, wherein the replacement amino acid residue atposition 299 is an alanine.
 56. The nucleic acid molecule of claim 54,wherein the replacement amino acid residue at position 299 is acysteine.
 57. The nucleic acid molecule of claim 54, wherein the aminoacid at position 299 is asparagine.
 58. The nucleic acid molecule ofclaim 54, wherein the amino acid at position 299 is glycine.
 59. Thenucleic acid molecule of claim 54, wherein the amino acid at position299 is tyrosine.
 60. The nucleic acid molecule of claim 54, wherein theamino acid at position 299 is histidine.
 61. The nucleic acid moleculeof claim 54, wherein the amino acid at position 299 is glutamic acid.62. The nucleic acid molecule of claim 54, wherein the amino acid atposition 299 is aspartic acid.
 63. The nucleic acid molecule of claim54, wherein the amino acid at position 299 is lysine.
 64. The nucleicacid molecule of claim 54, wherein the amino acid at position 299 isarginine.
 65. The nucleic acid molecule of claim 54, wherein the aminoacid at position 299 is isoleucine.
 66. The nucleic acid molecule ofclaim 54, wherein the amino acid at position 299 is leucine.
 67. Thenucleic acid molecule of claim 54, wherein the amino acid at position299 is methionine.
 68. The nucleic acid molecule of claim 54, whereinthe amino acid at position 299 is phenylalanine.
 69. The nucleic acidmolecule of claim 54, wherein the amino acid at position 299 is proline.70. The nucleic acid molecule of claim 54, wherein the amino acid atposition 299 is tryptophan.
 71. The nucleic acid molecule of claim 54,wherein the amino acid at position 299 is valine.
 72. A nucleic acidmolecule encoding a variant polypeptide of a parent polypeptidecomprising an IgG1 Fc region, wherein the Fc region of the variantpolypeptide comprises an amino acid at position 299 which differs fromthe amino acid at position 299 of the parent polypeptide, wherein theamino acid at position 299 of the parent polypeptide is threonine andthe amino acid at position 299 of the variant polypeptide is an alanine,and wherein the variant polypeptide comprises an amino acid at position297 of the Fc region having reduced glycosylation and displays reducedeffector function as compared to the parent polypeptide.
 73. A nucleicacid molecule encoding a variant polypeptide of a parent polypeptidecomprising an IgG4 Fc region, wherein the Fc region of the variantpolypeptide comprises an amino acid at position 299 which differs fromthe amino acid at position 299 of the parent polypeptide, wherein theamino acid at position 299 of the parent polypeptide is threonine andthe amino acid at position 299 of the variant polypeptide is an alanine,and wherein the variant polypeptide comprises an amino acid at position297 of the Fc region having reduced glycosylation and displays reducedeffector function as compared to the parent polypeptide.
 74. A vectorcomprising the nucleic acid of any one of claim 1, 10, 32, 54, 72 or 73.75. An isolated host cell comprising the nucleic acid of claim
 74. 76. Amethod for producing an antigen binding polypeptide comprising culturingthe host cell of claim 75 under conditions suitable for producing thepolypeptide by the host cell.
 77. A method of producing a modifiedantigen binding polypeptide having reduced glycosylation in an Fcregion, the method comprising, identifying a first amino acid residue atposition 299 in an original polypeptide and a second amino acid residueat position 297 capable of being glycosylated in an Fc region of theoriginal polypeptide wherein modification of the first amino acid willdecrease glycosylation at a second amino acid and modifying said firstamino acid residue to comprise said preferred side chain chemistry toproduce a modified polypeptide, wherein glycosylation of said secondamino acid residue of the Fc region is decreased in the modifiedpolypeptide as compared to the original polypeptide.